Evaluation of An I226R and A137R deletion mutant from a genotype I/II recombinant African swine fever virus strain as a live attenuated vaccine in pigs

Background: The emergence of highly lethal genotype I/II recombinant African swine fever virus (ASFV) strains in China has rendered existing genotype II-based live attenuated vaccines ineffective, underscoring the urgent need for novel vaccine candidates. Although single-gene deletions of A137R or I226R have shown promise against genotype II strains, their efficacy and safety in the context of recombinant strains remain unexplored. Methods: Using homologous recombination, we constructed a single gene-deleted mutant (JX23-02ΔI226R) and a double gene-deleted mutant (JX23-02ΔI226RΔA137R) from the genotype I/II recombinant ASFV strain JX23-02. The replication kinetics, pathogenicity, immunogenicity, and protective efficacy of these mutants were evaluated in vitro and in pigs. Results: Both deletion mutants exhibited significantly reduced replication in porcine alveolar macrophages. Immunization with JX23-02ΔI226R resulted in 40% survival, but surviving pigs were fully protected against subsequent lethal challenge. In contrast, JX23-02ΔI226RΔA137R was completely attenuated: all immunized pigs survived challenge without any clinical signs and developed robust p54-specific antibody responses. Moreover, viral shedding and tissue viral loads were markedly lower in the double-deletion group than in the single-deletion group. Conclusions: The double gene-deleted mutant JX23-02ΔI226RΔA137R represents a safe and efficacious live attenuated vaccine candidate against genotype I/II recombinant ASFV strains, highlighting the superiority of multi-gene deletion strategies for ASF vaccine development.